In the first cycle of SPORE funding, we developed a novel, reproducible, model for human superficial bladder cancer in athymic mice. With this model we demonstrated gene transfer and efficacy of intravesical interferon-a gene therapy when the adenoviral vector was administered with Syn3 (Ad-IFN-a/Syn3). We showed that Ad-IFN-a/Syn3 induced caspase-3 activation and apoptosis in some human bladder cancer cells that were insensitive to recombinant IFNa protein and that cell death occurred via direct and indirect (bystander) effects. Moreover, normal urothelial cells appear to be resistant to Ad-IFN-a/Syn3. Although the exact mechanisms underlying the effects of Ad-IFN-oc/Syn3 are unknown, the preclinical data were compelling enough to launch a Phase I study, which is scheduled to open in the fourth quarter of 2005. In this renewal we propose to define the unique mechanisms by which Ad-IFN-a mediates its antitumor activity. We also propose to develop pharmacodynamic urine-based markers that will allow us to track the biological activity of Ad-IFN-o/Syn3, focusing on the possibility that increased levels of soluble annexin 1 correlate with transduction efficiency, tumor cell apoptosis, or both. Finally, by using an elegant whole organ mapping technique developed by our colleagues in Project 1, we propose to accurately map the expression of interferon-a, in a proof-of-principle experiment that will follow the Phase I trial. To this end, we propose 3 Specific Aims. (1) To define the role of annexin-1 in apoptosis induced by Ad-IFN. (2) To identify the molecular mechanisms involved in the Ad-IFN-induced bystander effect. (3) To evaluate transgene expression of IFN-a and the activity of Ad-IFN/Syn3 following intravesical instillation into the bladder of patients with urothelial carcinoma These studies have the potential to establish a new paradigm for the therapy of superficial bladder cancer, which might qualitatively change the natural history of this disease.